HIV-1 Recombination In Natural Target Cells

描述（由申请人提供）：HIV-1的多样化和进化使该病毒能够持续存在，尽管有强烈的免疫应答和有效的抗逆转录病毒治疗。在全球范围内，病毒的多样化导致了不同的系统发育分支和亚型的公认的关注疫苗的发展。虽然大量的注意力一直致力于研究的病毒序列的变化所造成的连续单碱基取代，重组的贡献，HIV-1的多样化和进化是不太清楚。以前的研究定量逆转录病毒重组或检查影响重组的因素已经在体外使用纯化的病毒组分或转化的成纤维细胞进行，这些细胞不是体内感染的靶细胞。我们已经开发了允许快速和定量研究重组期间感染的基本上任何人类细胞类型，包括原代T细胞和巨噬细胞的方法。初步研究揭示：（1）通过检查相关靶细胞群中的HIV-1重组，揭示HIV-1比以前认识到的更具重组性，（2）HIV-1重组率在某些细胞类型中比在其它细胞类型中高得多，（3）单核细胞的分化导致重组率增加3倍，和（4）HIV-1重组可受逆转录酶内突变的影响，所述突变改变酶活性并赋予抗逆转录病毒药物临床抗性。基于这些初步研究，我们假设感染过程中的重组率与受感染细胞的分化、活化和/或增殖有关。我们还假设重组率直接与逆转录的动力学有关，逆转录的动力学由感染的细胞和RT中的突变决定，这会影响酶的功能。我们将通过以下具体目标来检验这些假设：1.确定髓系分化对重组率的影响。2.确定T细胞活化和增殖对重组的影响。3.确定目标1、2和4中逆转录和重组动力学之间的关系。4.确定影响RT活性和耐药性的突变在重组中的作用。

DESCRIPTION (provided by applicant): Diversification and evolution of HIV-1 enables the virus to persist despite a vigorous immune response and the administration of potent antiretroviral therapy. Globally, viral diversification has led to distinct phylogenetic clades and subtypes of recognized concern to vaccine development. While much attention has been devoted to the study of viral sequence changes resulting from sequential single base substitutions, the contribution of recombination to HIV-1 diversification and evolution is less well understood. Previous studies quantifying retroviral recombination or examining factors which affect recombination have been performed either in vitro using purified viral components, or with transformed fibroblastic cells that are not targets of infection in vivo. We have developed methods which permit rapid and quantitative study of recombination during infection of essentially any human cell type, including primary T cells and macrophages. Preliminary studies reveal (1) that by examining HIV-1 recombination in the relevant target cell population, HIV-1 is revealed to be more recombinogenic than previously appreciated, (2) that HIV-1 recombination rates are substantially higher in some cell types than in others, (3) that differentiation of monocytic cells leads to a 3 fold increase in recombination rates, and (4) that HIV-1 recombination can be influenced by mutations within the reverse transcriptase enzyme which alter enzyme activity and confer clinical resistance to antiretroviral drugs. Based on these preliminary studies, we hypothesize that the rate of recombination during infection is linked to the differentiation, activation and/or proliferation of the infected cell. We also hypothesize that recombination rates are directly linked to the kinetics of reverse transcription as determined by both the infected cell and by mutations in RT, which affect enzyme function. We will test these hypotheses through the following specific aims: 1. Determine the effects of myeloid differentiation on recombination rates. 2. Determine the effects of T cell activation and proliferation on recombination. 3. Determine the relationship between the kinetics of reverse transcription and recombination within aims 1, 2 and 4. 4. Determine the role in recombination of mutations which influence RT activity and drug resistance.